The ability of plants to be used as factories for the production of biopharmaceuticals has the potential to dramatically cut costs of production of many current biotherapeutics and will encourage development of new drugs and vaccines that can be genetically inserted into the genome of certain plants. Production via plants by growing biomass from plants that are genetically engineered to carry a specific biopharma product will allow for a fast and cost-effective multiplication of drugs that can be delivered to consumers at a substantial discount, and will allow numerous other advantages from current production methods. A significant roadblock to the production of plant-made-pharmaceuticals is the fact that plant materials containing a pharmaceutical gene could escape into the wild and genetically contaminate the food chain. To prevent escape of transgenic materials strict regulations have been put into effect by government agencies as a matter of oversight. In fact, it is not actually possible, nor is it practical to grow a crop of pharmaceutical plants in a farm production field, consequently, the need to produce pharmaceutical crops in fully controlled containment facilities that accommodate zero-tolerance regulations are now a necessity for entry into the plant biomanufacturing business and a biomanufacturing system to address these problems is a focus of this SBIR. In the current application we provide an example of a plant-made-pharmaceutical (recombinant hepatitis B vaccine) that we propose to take all the way from large scale controlled containment growth, secondary processing to provide stable shelf life and a human clinical trial in healthy adult volunteers. The award of the Phase I SBIR allowed us to establish the biomanufacturing baseline for measuring the antigen production levels of several HBsAg varieties of potato, for selection for highest antigen yield and increased production levels of the plant biomass. The plant production system being tested was developed by the SBIR Company, Quantum Tubers Corporation, and provides up to six crops per year in a computer controlled, modular system designed for biomanufacturing of potato plants. The QT Biomanufacturing System can provide as much as 36:1 production advantage over a seasonal greenhouse of the same size, and will provide exact and reproducible growing conditions to increase recombinant hepatitis B vaccine production in a GMP manufacturing environment. The Phase II SBIR application necessarily combines the logical path of progression for documentation of the QT Biomanufacturing System technology combined with the production of hepatitis B vaccine-bearing potatoes, verification of their vaccination potential through a Phase I Clinical Trial of the hepatitis B vaccine produced from the transgenic potato plant, and then the secondary processing of the raw biomass into a stable oral dose. The specific aims of the Phase II SBIR are : A) To quantify the economic production parameters providing for a best growing time vs. yield scenario for growing plant-derived hepatitis B vaccine using the QT Biomanufacturing System. B) To evaluate the safety, tolerability, and immunogenicity of orally delivered HBsAg that is formulated as an expressed protein in transgenic potato tubers (HBV-EPV) administered with a mucosal adjuvant to healthy adult subjects who have evidence of immunity to hepatitis B derived from previous immunization with licensed recombinant hepatitis B vaccine. C) To determine through R&D the most effective secondary processing system for potato leaf and tuber biomass to provide for the formulation of a shelf-stable, oral dosage of (HBsAg) hepatitis B vaccine and to evaluate in a preclinical model the in vivo immunogenicity of the processed material. The completion of this SBIR will bring together a combined set of technologies that will conclude years of research from multiple scientific disciplines that will now culminate in a next-generation set of technologies providing an alternative biotherapeutics production system for the pharmaceuticals industry. This SBIR will validate a new and market-changing technology that will accelerate production, lower delivered costs, provide a safer product, and bring vaccines and other biotherapeutics to the consumer. PUBLIC HEALTH RELEVANCE: More than 2 billion of the global population have been infected with hepatitis B virus estimated to cause 500,000 to 700,000 deaths each year worldwide. Plant-derived hepatitis B vaccine from potatoes grown in controlled environment biomanufacturing facilities will provide for orally administered vaccines that can be manufactured as large quantities of shelf-stable, non-refrigerated, oral vaccines that can dramatically lower global deaths and infections from the hepatitis B virus because of affordability of the vaccination. Our production system would revolutionize worldwide vaccine delivery along with other health programs where biotherapeutics could be employed. Oral vaccination would eliminate many of the problems associated with the current needle delivery method for vaccines including refrigeration ('cold chain" requirements,) storage & handling limitations for liquid vaccines along with the need for hypodermic needles that are regularly reused in third world countries resulting in the spread of hepatitis B and even HIV/AIDS from previously infected individuals. Oral vaccines are not only easier to administer than injected vaccines, allow for compliance in children and adults but they also stimulate both systemic and mucosal immune responses, whereas injected vaccines only lead to serum antibody production. Stimulating an immune response at the mucosal sites (such as the nose and mouth) is very desirable, because many pathogens enter the body at these sites. If an immune response occurs at the mucosal sites, pathogens can be prevented from even entering the body. The overall goals of this project are to document a biomanufacturing system, and to validate a vaccine produced by plants delivered to humans as part of a clinical trial, thus creating an integrated plant-derived vaccines delivery system for worldwide deployment. [unreadable] [unreadable] [unreadable]